Treatment of immune disorders with Hom-1 inhibitors

ABSTRACT

Disclosed are methods for treating immune disorders using inhibitors of Hom-1. The inhibitors include an RNAi agent containing the sequence of SEQ ID NO: 6.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/US10/42126, filed on Jul. 15, 2010, which claims the priority ofU.S. Provisional Application No. 61/225,852, filed on Jul. 15, 2009. Thecontents of both applications are hereby incorporated by reference intheir entirety.

BACKGROUND

The immune system defends the human body against pathogen infection,cellular transformation, and physical/chemical damage. Its dysfunction,either overactive or underactive, leads to various disorders. Thedysfunction can be caused by aging, development defects, diseases, andmedical treatment (e.g., chemotherapy or immuno-suppression). There is aneed for drugs and reagents for treatment and diagnosis of immunedisorders.

SUMMARY

This invention relates to materials and methods for treatment anddiagnosis of immune disorders using Hom-1 or its inhibitors oractivators. Shown below are the polypeptide and nucleotide sequences ofHom-1.

Hom-1 polypeptide (SEQ ID NO: 1):mrlssspprg pqqlssfgsv dwlsqsscsg pthtprpadf slgslpgpgq tsgareppqavsikeaagss nlpapertma glskepntlr aprvrtaftm eqvrtlegvf qhhqylsplerkrlaremql sevqiktwfq nrrmkhkrqm qdpqlhspfs gslhappafy stssglanglqllcpwapls gpqalmlppg sfwglcqvaq ealasagasc cgqplashpp tpgrpslgpalstgprglca mpqtgdaf (Underlined: aa. 91-151/homeodomain, SEQ ID NO: 5)Hom-1 nucleotide sequence (SEQ ID NO: 9):acctggccgc catgcgcctc tcctcctccc cacctcgtgg cccgcagcag ctctccagctttggctccgt ggactggctc tcccagagca gctgctcagg gccgacccac acccccaggcctgccgactt ctccctgggg agcctccctg gcccaggcca gacatccggc gcccgggagccccctcaggc cgtcagcatc aaggaggccg ccgggtcctc aaatctgcct gcgccggagaggaccatggc cgggttgagt aaggagccaa ataccttgcg ggccccccgt gtccgcacagccttcaccat ggagcaggtc cgcaccttgg agggcgtctt ccagcaccac cagtacctgagccctctgga gcggaagagg ctggccaggg agatgcagct ctcagaggtc cagataaaaacctggtttca gaatcgccgc atgaaacaca aacggcaaat gcaggacccc cagctgcacagccccttctc ggggtctctc catgcgcccc cagctttcta ctcaacgtct tctggccttgccaatggcct gcagctgctg tgcccttggg cacccctgtc cgggccccag gctctgatgctgccccctgg ctccttctgg ggtctctgcc aagtggcaca agaggccctg gcatctgcgggagcttcctg ctgcgggcag cctctggcgt cccacccccc taccccaggc cggccttcgctgggaccagc cctgtccacg gggccccggg gcctgtgtgc tatgccacag acgggggatgcattttgagg aggcacctct gactcccaca ctcgcggtct tgctgatcgc acctggctcctacctggagg actcagttgt tctgtttaca tcctggtggc acctctcacc ctgacccacacaaaggttct ggagattact ggagaatata tataaatata tatatgtacg tatatatgtaaatacacata tacgtatata taaatatata tatacatatg tgtgtgtata tatatatatatttttttttt tttttttttt tttgagacgg agtgttgctc tgtcacccag gctggagtgcaatgacgcaa tctcggctca ctgcaacctc cgcctcctgg gttcaagcga ttctccagcctcagcctccc gagtagctgg gattacagac acccgccacc acgcccggct aattttttctatttttagta gaaatggggt ttcaccatgt tagccaggct ggtctcaaac tcctgaccctgtgatccgcc cgcctcggcc tcccaaagtg ctgggattac aggcatgagc cactgcacccggccctgaga atatatttat taaagccacc tcttcactga aagttaccga aagagtcggtttaggaagga aacgaagggt cagtgaacag agtcaaatgc agaagtgggc ttgtcatgggtagggctttc ggcgtacgat aaaaggatca tttgtttttt aaaaggggtt ggaaaaactggttttccagt tggaaacagt aaaggttgta agctttgtgt gtacaaaaga aaacagggaatgcaggtgtg tttatagcgt tgtggttcaa gtccctctta acaagaactc caaagctggaaagcaggagg gaacaaaggt gaacatgaag gcgaggatgc tggggccctg cagtgcgctctaggctgtgc gtgagccggg actgtaccca cagcttgctg agggctgctc ttcttgggccagggaaagca gggcagccgg gacctgcggc tgtgcctgga ctgaagctgt cccgcaggtccccaccctcc aacacgtgct cacctgtccc cctcctcgca gcagcctcgg gacaaaacaatgactcaagg acagcacttc tcgcagaagg tctggaagtg cccagaatgg gaggcacggaagcccctccc ggggaggact cccgcgttga tggaccgttc ttggtgcaga ctcctgactgcgtgcatgaa acctgagaca agtgcaattc cttccatgtc gccccagagt gcccaggaggcaggcagtgc ggggtgccca ggcagacggg ttcagcctgc agaactggag gcgacctgtgaaacccaccc gggcacccca acaggaacag aagcgtggtc ctgcggctgc gtccccagcgagtttcactt tccccttgct cgtttctccc ttgttgtaag tgtttacaac tggcatgtgcttttaaacgt caggtaagag gggaacagct gctgtacatc gtcctggcga gtgacaatgtgacagaagcc tgggcgaggc cctcggaggg cagcagctgg acaggggcta ctgggtttggcctggacagc actgatttgt ggatgtggat gggggcacgt tgtccgtgat aaaagtacaagtgcccctca caaaaaaaaa aaaaaaaa(underlined: coding sequence (nt12 to 788, SEQ ID NO: 2)

Accordingly, in one aspect, the invention features an RNAi agent havinga first strand having a first nucleotide sequence homologous to a regionof a gene encoding Hom-1 protein. The RNAi agent targets an mRNAtranscribed from the gene or its 5′ un-translated area. In one example,the first sequence includes UUCAGAAUCGCCGCAUGAAACACAAACGG (SEQ ID NO:6), UCUACUCAACGUCUUCUGGCCUUGCCAAU (SEQ ID NO: 7), and the correspondingDNA version: TTCAGAATCGCCGCATGAAAC ACAAACGG (SEQ ID NO: 4),TCTACTCAACGTCTTCTGGCCTTGCCAAT (SEQ ID NO: 8), The RNAi agent can alsoinclude a second strand having a second nucleotide sequencecomplementary to the first sequence.

The invention also features a pharmaceutical composition having thejust-mentioned RNAi agent. The composition can be a nasal aerosol or aninhalation composition.

The above-described agent and composition can be used for treating ahuman subject having, or at risk of having, an immune disorder, such asan inflammation disorder, including acute respiratory distress syndrome(ARDS), which can be caused by virus infection or chemicals.Specifically, one can administer to a subject in need thereof aneffective amount of the RNAi agent or composition. In one embodiment,the RNAi agent has the nucleotide sequence of SEQ ID NO: 6 or 7. Thesubject can be one that has, or is suspected of having, an influenza,e.g., SARS.

In a second aspect, the invention features an isolated nucleic acidhaving SEQ ID NO: 4, 6, 7, or 8 mentioned above or its complement. Theinvention also features an isolated nucleic acid having the sequencelisted below (SEQ ID NO: 3) or its complement.

5′cgaatgcagaggctcctgcgatggccccggagtgagtcccccagaggagccggattagggctggaggcggccgagtcccccgagaggcccctcccgacattcccgcccccgcgcgccgctccccgggtcctccgcgtctctttcccgggaaagcctccctcggttcctgcgcggccgcacagcctggacgcagcgcacgcgggcaccggcctgactctcccaccccgaagcctgctcccaacctaagtccgccctgactctcccagcctgaagcctgctcgccctcgggtgtccgggctgggcacaggcgccagcgtccccctggagaggagaggtcgcccggcacctcccaggacaggcccaagtgggagtgggaccctcctaccttcctgcagcctcggcccgcggggtggggggttgggagagatgaaaggaggtgaccgatcccgaaccatcgcctctccattaaccagggcccgcagccccgcccctcccccagacatcgaggagccggggaggtgtgaacggcctcctttgtgcctctgaatcgaaggcaattaggcgctgcttatctgggcattagccgtgtatgcaaaccgggctcccgccccctcctcctgggcttataaacgccgccgcctggcgaggcccgaggtggatcctgcgcctggccagccccgcctggccttccctccggcccacctggccgcc3′

In a third aspect, this invention features a method for decreasing thelevel or activity of inflammatory cells (e.g., macrophages) in asubject. The method includes administering to a subject in need thereofan effective amount of an inhibitor of a polypeptide containing thesequence of SEQ ID NO: 1.

In a fourth aspect, this invention features a method for decreasing thelevel of a pro-inflammatory cytokine in a subject by administering to asubject in need thereof an effective amount of an inhibitor of apolypeptide containing the sequence of SEQ ID NO:1. Examples of thecytokine include TNF-α, IL-1β, and IL6.

In a fifth aspect, this invention features a method for treating a humansubject having, or at risk of having, an immune disorder. The methodincludes administering to a subject in need thereof an effective amountof an inhibitor of a polypeptide containing the sequence of SEQ IDNO: 1. The immune disorder can be an inflammatory or autoimmunedisorder, such as ARDS. In one example, the subject has or is suspectedof having influenza.

In the above-mentioned methods, examples of the inhibitor include anantibody (such as antibody that leads to cell growth, includinganti-CD3), an antisense nucleic acid, and an RNAi agent, as well asother macro molecule or small molecule compounds and naturally occurringcompounds, which target Hom-1. The RNAi agent can have the nucleotidesequence of SEQ ID NO: 4, 6, 7, or 8. Examples of small moleculecompounds include Prednisone, Imuran, methrotrexate, cellcept, andionomycin, Examples of macro molecule includes PHA. In one example, eachof the method further includes determining the level of Hom-1'sexpression or activity in a sample obtained from the subject before orafter the administration so as to confirm inhibition of Hom-1.

In a sixth aspect, this invention features a method for increasing thelevel or activity of inflammatory cells (e.g., macrophages) in asubject. The method includes administering to a subject in need thereofan effective amount of a polypeptide containing the sequence of SEQ IDNO: 1 or 5, or a functional equivalent thereof, or a nucleic acidencoding the polypeptide, or an activator of Hom-1. Examples of theactivator include 5-FU, DOX, radiation, retinoic acid, GM-CSF-IL4,resveratrol, ellagic acid, aspirin, salicylic acid, emodin and flavonoidand their derivatives that induce Hom-1 expression. In one example, themethod further includes determining the level of Hom-1's expression oractivity in a sample obtained from the subject before or after theadministration so as to confirm induction of Hom-1.

A functional equivalent refers to a polypeptide that is similar to, oris a derivative of, a common polypeptide, e.g., a protein having one ormore point mutations, insertions, deletions, truncations, a fusionprotein, or a combination thereof, and retaining substantially theability of the common polypeptide, such as binding to a LEF1/TCF. In oneexample, the polypeptide lacks an LEF1/TCF transcactivation domain. Thecellular proliferative disorder can be a condition characterized byaberrant activation of LEF1/TCF-mediated transcription. An aberrantactivation of LEF1/TCF-mediated transcription refers to a cellularcondition where the LEF1/TCF-mediated transcription is abnormally high,as determined by the assays described in the example below or anyanalogous assays. Example of SEQ ID NO: 5's functional equivalentinclude the homeodomain sequences of Xenopus (i.e., Xom), chimpanzee,and rhesus. These homeodomain sequences are listed in FIG. 1. Ingeneral, they are at least 30% (e.g., 40, 50, 60, 65, 70, 75, 80, 85,90, or 95%) identical to SEQ ID NO: 5. For example, the homeodomain ofHom-1 shares 68% identical and 85% positive (similar) amino acidsequence with Xom

In a seventh aspect, the above-described nucleic acids can be used in amethod of diagnosing an immune disorder, including (1) cellularproliferative disorder/cancer and (2) inflammatory or autoimmunedisorder. Examples of the cellular proliferative disorders include acutelymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), andacute myloid leukemia (AML). Examples of the inflammatory or autoimmunedisorder include Myelodysplastic syndromes (MDS), Systemic LupusErythematosus (SLE), inflammatory bowel diseases (IBD), rheumatoidarthritis (RA), and transplant rejection. The method includes the stepof obtaining a biological sample from the subject; and determining theexpression level of a gene encoding a polypeptide containing SEQ ID NO:1 in the sample. The subject is determined to have or be prone todevelop cellular proliferative disorder/cancer (ALL, CLL, and AML) ifthe expression level is below a first predetermined level or absent. Thesubject is determined to have or be prone to develop an inflammatory orautoimmune disorder if the expression level is above a secondpredetermined level. Such a predetermined level can be obtained from anormal control subject.

In an eighth aspect, the invention further features a method formanaging patient treatment. The method includes steps of identifying apatient under, or in need of, a treatment for a condition, obtaining abiological sample from the patient, and determining the expression levelof a gene encoding a polypeptide containing SEQ ID NO: 1 in the sample.The patient is determined to be suitable for the treatment if the levelis at or above a predetermined value. Alternatively, if the level islower than that level, the patient should not be subject to thetreatment. The method can further include a step of communicating theexpression level to the patient or to a physician or a caretaker of thepatient. In one example, the condition is a cellular proliferativedisorder, or an immune disorder, such as the above-mentioned ALL, CLL,AML, and MDS.

For ALL, CLL, AML or MDS, where the expression level of Hom-1 is lowerthan a predetermined value, Hom-1 can serve as a marker to direct thechoice of effective treatment strategy; for transplant patients, Hom-1can be used to ensure adequate amount of immune-suppressant is used; forauto-immune diseases, such as SLE, Hom-1 can be used to monitor the useof immune-suppressant. For example, if Hom-1 expression level decreasesto a said value upon the using of immuno-suppressants, it will indicatethe need to stop the usage of the immune-suppressants to avoidlymphoproliferative disorder, such as lymphoma.

In a ninth aspect, the invention features a kit for diagnosing theabove-mentioned disorders or for managing patient treatment. The kitincludes one or more reagents selected from the group consisting of: anantibody specific for a polypeptide having the sequence of SEQ ID NO: 1,a polypeptide having the sequence of SEQ ID NO: 1, a pair of PCR primersfor amplifying a fragment of SEQ ID NO: 2 or 3, and a nucleic acid that,under a stringent condition, hybridizes to the compliment of a referencenucleic acid, wherein the reference nucleic acid consists of SEQ ID NO:2, 3, or 5.

Stringent hybridization conditions can be suitably selected by oneskilled in the art, and for example, low-stringent conditions can begiven. Low-stringent conditions are, for example, 42° C., 2×SSC, and0.1% SDS, and preferably, 50° C., 2×SSC, and 0.1% SDS. Highly stringentconditions are more preferable and include, for example, 65° C., 2×SSC,and 0.1% SDS. However, several factors other than temperature, such assalt concentration, can influence the stringency of hybridization andone skilled in the art can suitably select the factors to accomplish asimilar stringency.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B are amino acid sequence alignments. FIG. 1A shows analignment between Xom and Hom-1 homeodomain-containing regions (SEQ IDNO: 19 and residues 93-152 of SEQ ID NO:1, respectively). FIG. 1B showsan amino acid sequence alignment of Hom-1 (SEQ ID NO:1), predictedchimpanzee (SEQ ID NO: 20) and monkey Hom-1 (SEQ ID NO: 21) homologues,indicating that the Hom-1 sequence is conserved in primates.

FIGS. 2A and 2B are a schematic presentation of Hom-1 and its deletionmutants and their in vivo interaction with TCF4, determined byimmunoprecipitation western blot analysis (FIG. 2A) and photographsshowing co-localization of Hom-1 and TCF4 in HCT116 cells (FIG. 2B).

FIGS. 3A-3D are diagrams showing results of: (A) cell viability asdetermined by trypan blue staining (FIG. 3A, left panel), MTS assay(FIG. 3A, middle panel), and ³H-thymidine incorporation assay (FIG. 3A,right panel); (B) down-regulation of Hom-1 expression with Hom-1 shRNA(FIG. 3B); (C) effects of down-regulation of Hom-1 on the proliferationof Nalm16 as measured by MTT assay (FIG. 3C, upper panel) and cellviability count (FIG. 3C, lower panel; (D) down-regulation of Hom-1,which led to elevated expression of cyclin D1, with the expression levelof tubulin as an internal control (FIG. 3D).

FIGS. 4A-4D are photographs or a diagram (FIG. 4C, right panel) showingresults of: (A) a tissue expression profiling of Hom-1 expression inadult tissues, using RT-PCR with GAPDH as internal control; (B) alineage analysis showing that Hom-1 is expressed mainly in cells derivedfrom both the myeloid and lymphoid lineages, including monocytes, Tcells, B cells, and neutrophils; (C) Hom-1's increasing expressionduring B cell development; (D) Hom-1 expression study showing that Hom-1is not expressed in most cancer cells derived from B-cell malignancies,except Nalm16.

FIGS. 5A and 5B are (A) a photograph showing that Hom-1 expression wasmarkedly reduced in peripheral blood samples from newly diagnosed tenCLL patients (FIG. 5A) and (B) a diagram showing that down-regulation ofHom-1 in lymphocytic leukemia was associated with correspondingelevation in the expression of cyclin D1 with GAPDH used as internalcontrol (FIG. 5A).

FIGS. 6A-6D are diagrams (FIGS. 6A and 6C) and photographs (FIGS. 6B and6D) showing: (A) transfection rates of wild type and p53 knockdownHCT116 cells transfected with constructs encoding GFP or GFP-Hom-1 (FIG.6A); (B) effects of Hom-1 on tumor-bearing nude mice (FIG. 6B) and tumorvolumes (FIG. 6C), where tumor growth was significantly repressed inHom-1-transfected p53 WT and KO cell lines. (Values are mean±SD, n=5 pergroup, *P<0.01 v.s. vector-transfected cells; (C) apoptosis in xenografttumors tissue as examined by TUNEL staining (FIG. 6D).

DETAILED DESCRIPTION

This invention is based, at least in part, on the unexpected discoveriesof that Hom-1 or its inhibitors can be used for treating or diagnosingvarious immune disorders.

As described herein, Hom-1 is a LEF/TCF-associated factor that repressescanonical Wnt/beta-catenin signaling by disrupting the formation of thebeta-catenin/LEF/TCF complex. Gain of function and loss of functionapproaches defined Hom-1 as a negative regulator of cell growth. Hom-1is highly expressed in normal hematopoietic cells (and Hom-1 expressionis up-regulated during maturation of hematopoietic cells), but itsexpression is significantly reduced in human lymphocytic leukemia andduring immortalization of peripheral B lymphocytes by EBV infection.Altered expression of Hom-1 is associated with corresponding changes ofthe Wnt/beta-catenin/LEF/TCF target oncogenes, such as cyclin D1,suggesting Hom-1's role in the pathogenesis of various immune disordersand hematological malignancies. Accordingly, this invention featuresmaterials and methods for treating or diagnosing these disorders.

For example, a polynucleotide containing a nucleic acid sequenceencoding an inhibitor of Hom-1 can be used to treat aninflammation-related disorder, such as ARDS. This inhibitor can be usedas immune suppressant. The nucleic acid sequence can encode a smallinterference RNA (e.g., an RNAi agent) that targets Hom-1 and inhibitsits expression or activity.

A nucleic acid refers to a DNA molecule (e.g., a cDNA or genomic DNA),an RNA molecule (e.g., an mRNA), or a DNA or RNA analog. A DNA or RNAanalog can be synthesized from nucleotide analogs. The nucleic acidmolecule can be single-stranded or double-stranded, but preferably isdouble-stranded DNA. An “isolated nucleic acid” is a nucleic acid thestructure of which is not identical to that of any naturally occurringnucleic acid or to that of any fragment of a naturally occurring genomicnucleic acid. The term therefore covers, for example, (a) a DNA whichhas the sequence of part of a naturally occurring genomic DNA moleculebut is not flanked by both of the coding sequences that flank that partof the molecule in the genome of the organism in which it naturallyoccurs; (b) a nucleic acid incorporated into a vector or into thegenomic DNA of a prokaryote or eukaryote in a manner such that theresulting molecule is not identical to any naturally occurring vector orgenomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment,a fragment produced by polymerase chain reaction (PCR), or a restrictionfragment; and (d) a recombinant nucleotide sequence that is part of ahybrid gene, i.e., a gene encoding a fusion protein.

The term “RNAi agent” refers to an RNA (or analog thereof), havingsufficient sequence complementarity to a target RNA to direct RNAinterference. Examples also include a DNA that can be used to make theRNA. RNA interference (RNAi) refers to a sequence-specific or selectiveprocess by which a target molecule (e.g., a target gene, protein or RNA)is down-regulated. Generally, an interfering RNA (“iRNA”) is a doublestranded short-interfering RNA (siRNA), short hairpin RNA (shRNA), orsingle-stranded micro-RNA (miRNA) that results in catalytic degradationof specific mRNAs, and can also be used to lower or inhibit geneexpression.

Thus, also within the scope of this invention is utilization of RNAifeaturing degradation of RNA molecules (e.g., within a cell).Degradation is catalyzed by an enzymatic, RNA-induced silencing complex(RISC). A RNA agent having a “sequence sufficiently complementary to atarget RNA sequence, e.g., Hom-1, to direct RNAi” means that the RNAagent has a sequence sufficient to trigger the destruction of the targetRNA by the RNAi machinery (e.g., the RISC complex) or process. A RNAagent having a “sequence sufficiently complementary to a target RNAsequence to direct RNAi” also means that the RNA agent has a sequencesufficient to trigger the translational inhibition of the target RNA bythe RNAi machinery or process. A RNA agent can also have a sequencesufficiently complementary to a target RNA encoded by the target DNAsequence such that the target DNA sequence is chromatically silenced. Inother words, the RNA agent has a sequence sufficient to inducetranscriptional gene silencing, e.g., to down-modulate gene expressionat or near the target DNA sequence, e.g., by inducing chromatinstructural changes at or near the target DNA sequence. The term “RNA” or“RNA molecule” or “ribonucleic acid molecule” refers to a polymer ofribonucleotides. The term “DNA” or “DNA molecule” or “deoxyribonucleicacid molecule” refers to a polymer of deoxyribonucleotides. DNA and RNAcan be synthesized naturally (e.g., by DNA replication or transcriptionof DNA, respectively). RNA can be post-transcriptionally modified. DNAand RNA can also be chemically synthesized. DNA and RNA can besingle-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded(e.g., double-stranded, i.e., dsRNA and dsDNA, respectively).

The above-mentioned polynucleotides can be delivered using polymeric,biodegradable microparticle or microcapsule delivery devices known inthe art. Another way to achieve uptake of the polynucleotides is usingliposomes, prepared by standard methods. The polynucleotide can beincorporated alone into these delivery vehicles or co-incorporated withtissue-specific antibodies. Alternatively, one can prepare a molecularconjugate composed of a plasmid or other vector attached topoly-L-lysine by electrostatic or covalent forces. Poly-L-lysine bindsto a ligand that can bind to a receptor on target cells (Cristiano, etal., 1995, J. Mol. Med. 73:479). Alternatively, tissue specifictargeting can be achieved by the use of tissue-specific transcriptionalregulatory elements that are known in the art. Delivery of naked DNA(i.e., without a delivery vehicle) to an intramuscular, intradermal, orsubcutaneous site is another means to achieve in vivo expression.

In the above-mentioned polynucleotides, e.g., expression vectors, thenucleic acid sequence encoding an inhibitor of Hom-1 is operativelylinked to a promoter or enhancer-promoter combination. Suitableexpression vectors include plasmids and viral vectors such as herpesviruses, retroviruses, vaccinia viruses, attenuated vaccinia viruses,canary pox viruses, adenoviruses and adeno-associated viruses.

SiRNA, miRNA, and asRNA (antisense RNA) molecules can be designed bymethods well known in the art. SiRNA, miRNA, and asRNA molecules withhomology sufficient to provide sequence specificity required to uniquelydegrade any RNA can be designed using programs known in the art,including those maintained on websites for Ambion, Inc. and Dharmacon,Inc. Systematic testing of several designed species for optimization ofthe siRNA, miRNA, and asRNA sequence can be routinely performed by thoseskilled in the art. Considerations when designing short interferingnucleic acid molecules include biophysical, thermodynamic, andstructural considerations, base preferences at specific positions in thesense strand, and homology. These considerations are well known in theart and provide guidelines for designing the above-mentioned RNAmolecules for, e.g., intranasal delivery to the lungs as described inthe present application.

In one aspect, the above-described agents or a composition containingthe agents can be used to treat an inflammation-related disorder in asubject. An inflammatory or inflammation-related disorder ischaracterized by a local or systemic, acute or chronic inflammation.Examples include inflammatory dermatoses (e.g., dermatitis, eczema,atopic dermatitis, allergic contact dermatitis, urticaria, necrotizingvasculitis, cutaneous vasculitis, hypersensitivity vasculitis,eosinophilic myositis, polymyositis, dermatomyositis, and eosinophilicfasciitis), inflammatory bowel diseases (e.g., Crohn's disease andulcerative colitis), acute respiratory distress syndrome, fulminanthepatitis, pancreatitis, hypersensitivity lung diseases (e.g.,hypersensitivity pneumonitis, eosinophilic pneumonia, delayed-typehypersensitivity, interstitial lung disease or ILD, idiopathic pulmonaryfibrosis, and ILD associated with rheumatoid arthritis), asthma, andallergic rhinitis. Examples also include autoimmune diseases (e.g.,rheumatoid arthritis, psoriatic arthritis, systemic lupus erythematosus,myasthenia gravis, juvenile onset diabetes, glomerulonephritis,autoimmune throiditis, ankylosing spondylitis, systemic sclerosis, andmultiple sclerosis), acute and chronic inflammatory diseases (e.g.,systemic anaphylaxia or hypersensitivity responses, drug allergies,insect sting allergies, allograft rejection, and graft-versus-hostdisease), Sjogren's syndrome, human immunodeficiency, and virusinfection.

In another aspect, Hom-1 over-expression and its activators can be usedto enhance immunity so as to treat cellular proliferative disorder, suchas cancer (e.g., brain, breast, prostate, colon, kidney, ovary, thyroid,lung, and hematopoietic cancer), and tumor metastasis.

A “subject” refers to a human and a non-human animal. Examples of anon-human animal include all vertebrates, e.g., mammals, such asnon-human primates (particularly higher primates), dog, rodent (e.g.,mouse or rat), guinea pig, cat, and non-mammals, such as birds,amphibians, reptiles, etc. In a preferred embodiment, the subject is ahuman. In another embodiment, the subject is an experimental animal oranimal suitable as a disease model. “Treating” or “treatment” refers toadministration of a compound or agent to a subject, who has a disorder(e.g., ARDS or similar respiratory tract/lung disorders), with thepurpose to cure, alleviate, relieve, remedy, delay the onset of, orameliorate the disorder, the symptom of the disorder, the disease statesecondary to the disorder, or the predisposition toward the disorder. An“effective amount” refers to an amount of the compound that is capableof producing a medically desirable result, e.g., as described above, ina treated subject. The treatment method can be performed in vivo or exvivo, alone or in conjunction with other drugs or therapy.

A composition of this invention can be administered parenterally,orally, nasally, rectally, topically, or buccally. The term “parenteral”as used herein refers to subcutaneous, intracutaneous, intravenous,intrmuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional, or intracranial injection, aswell as any suitable infusion technique.

In one embodiment, the above-described composition can be used to treatARDS, and similar or related respiratory tract/lung disorders. Examplesinclude a respiratory tract or lung infection, which refers to anybacterial, viral, fungal, or parasite infection of any part of therespiratory system.

The nucleic acid molecules can be prepared in any aqueous carrier,vehicle, or solution so as to provide a composition that ispharmaceutically suitable for in vivo administration. Methods ofpreparing aqueous solutions are well known to one of ordinary skill inthe art. Preferably, the aqueous solutions is water, physiologicallyacceptable aqueous solutions containing salts and/or buffers, such asphosphate buffered saline (PBS), or any other aqueoussolution/surfactant acceptable for administration to a animal or human.Such solutions are well known to a person skilled in the art andinclude, but are not limited to, distilled water, de-ionized water, pureor ultrapure water, saline, PBS, and solutions containing usual bufferswhich are compatible with nucleic acids. The compositions may alsocontain sodium chloride and glucose or mannitol to make the solutionisotonic. The composition may contain suitable auxiliary components suchas pH, osmolarity and tonicity adjusting agents.

For administration via the upper respiratory tract, the composition isformulated into a solution, e.g., water or isotonic saline, buffered orunbuffered, or as a suspension, at an appropriate concentration forintranasal administration as drops or as a spray. Preferably, suchsolutions or suspensions are isotonic relative to nasal secretions andof about the same pH, ranging e.g., from about pH 4.0 to 7.4 or, from pH6.0 to 7.0. Buffers should be physiologically compatible and include,simply by way of example, phosphate buffers. For example, arepresentative nasal decongestant is described as being buffered to a pHof about 6.2 (Remington's Pharmaceutical Sciences 16th edition, Ed.Arthur Osol, page 1445 (1980)). One skilled in the art can readilydetermine a suitable saline content and pH for an innocuous aqueoussolution for nasal and/or upper respiratory administration. Othersuitable aqueous vehicles include, but are not limited to, Ringer'ssolution and isotonic sodium chloride. Aqueous suspensions may includesuspending agents such as cellulose derivatives, sodium alginate,polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such aslecithin. Suitable preservatives for aqueous suspensions include ethyland n-propyl p-hydroxybenzoate.

The compositions may contain minor amounts of polymers, surfactants, orother excipients well known to those of the art. In this context, “minoramounts” means no auxiliary agents or substances are present that mightaffect or mediate uptake of nucleic acid in the cells of the lungs.

Aerosol dosage, formulations and delivery systems may be selected for aparticular therapeutic application, as described, for example, in Gonda,Critical Reviews in Therapeutic Drug Carrier Systems, 6:273-313, 1990.The term aerosol as used herein refers to any preparation of a fine mistof particles, which can be in solution or a suspension, whether or notit is produced using a propellant. Aerosols can be produced usingstandard techniques, such as ultrasonication or high pressure treatment.

The formulation may be administered in an aqueous solution that ispharmaceutically acceptable for administration to the respiratorysystem. Particle sizes greater than 5 μm are deposited in the nasalcavity. Particles that are 2 to 10 μm can be retained in the lungs, andparticles of less than 1 μm are exhaled. In preferred embodiments, thecompound is administered through inhalation in a form such as liquidparticles and/or solid particles. Suitable examples include, but are notlimited to, an aerosol, a nebula, a mist, an atomized sample, and liquiddrops. Typical apparatus which may be used for administration to humansinclude metered dose inhalers (MDI), nebulizers, and instillationtechniques. The formulation is administered in an amount effective totreat, prevent, or diagnose on one or more symptoms or manifestations oflung disease. It is believed that the nucleic acid molecules can also beadministered as dry powders using a dry powder inhaler, where theparticles dissolve within the lung secretions. Various suitable devicesand methods of inhalation which can be used to administer particles to apatient's respiratory tract are known in the art. Nebulizers create afine mist from a solution or suspension, which is inhaled by thepatient. See, e.g., U.S. Pat. No. 5,709,202.

The compositions are preferably delivered into the lung with apharmacokinetic profile that results in the delivery of an effectivedose of the nucleic acid. As generally used herein, an “effectiveamount” of a nucleic acid of the invention is that amount which is ableto treat one or more symptoms of a lung disease, reverse the progressionof one or more symptoms of a lung disease, halt the progression of oneor more symptoms of a lung disease, prevent the occurrence of one ormore symptoms of a lung disease, decrease a manifestation of the diseaseor diagnose one or more symptoms of a lung disease in a subject to whomthe compound or therapeutic agent is administered, as compared to amatched subject not receiving the compound or therapeutic agent. Theactual effective amounts of drug can vary according to the specific drugor combination thereof being utilized, the particular compositionformulated, the mode of administration, and the age, weight, conditionof the patient, and severity of the symptoms or condition being treated.Dosages for a particular patient can be determined by one of ordinaryskill in the art using conventional considerations, (e.g. by means of anappropriate, conventional pharmacological protocol). In one embodiment,the compositions are delivered at a dose range of 3 to 400 μg per 20 gof body weight, with upper dosing limit of 1 gram per 20 grams bodyweight. In a preferred embodiment the compositions are delivered at adose range of 50 to 100 μg per 20 g of body weight. In another preferredembodiment the compositions are delivered at a dose range of 150 nM perkg of body weight.

One or more of the above-described agents can be administered to ananimal (e.g., a human) to modulate expression or activity of Hom-1 orits homologus. A physician may, for example, prescribe a relatively lowdose at first, subsequently increasing the dose until an appropriateresponse is obtained. In addition, it is understood that the specificdose level for any particular subject will depend upon a variety offactors including the activity of the specific compound employed, theage, body weight, general health, gender, and diet of the subject, thetime of administration, the route of administration, the rate ofexcretion, any drug combination, and the degree of expression oractivity to be modulated.

The efficacy of treatment can be monitored either by measuring theamount of the target gene mRNA (e.g. using real time PCR) or the amountof polypeptide encoded by the target gene mRNA (Western blot analysis).As is well known in the art, the dosage for a patient depends uponvarious factors as described above. Dosages will vary, but a preferreddosage for administration of polynucleotide is about 10⁶ to 10¹² copiesof the polynucleotide molecule. This dose can be repeatedly administeredas needed. Routes of administration can be any of those listed above.

This invention also features diagnosis methods. A cancer cell or a cellprone to tumorigenesis can be detected in a subject based on the absenceof the Hom polypeptide (e.g., antibody) or a nucleic acid (e.g., genomicDNA or mRNA) encoding the polypeptide in a test sample from the subject.In other words, the polypeptide and nucleic acids can be used as markersto indicate the presence or absence of a cancer cell. Diagnostic andprognostic assays of the invention include methods for assessing theexpression level of the Hom polypeptide or nucleic acid and foridentifying variations and mutations in the sequence of the Hompolypeptide or nucleic acid.

The presence, level, or absence of the Hom polypeptide or nucleic acidin a test sample can be evaluated by obtaining a test sample from a testsubject and contacting the test sample with a compound or an agentcapable of detecting the Hom polypeptide or nucleic acid (e.g., mRNA orgenomic DNA probe). The “test sample” includes tissues, cells andbiological fluids isolated from a subject, as well as tissues, cells andfluids present within a subject. The level of expression of the Hom genecan be measured in a number of ways, including measuring the mRNAencoded by the Hom gene; measuring the amount of polypeptide encoded bythe Hom gene; or measuring the activity of polypeptide encoded by theHom gene.

The level of mRNA corresponding to the Hom gene in a cell can bedetermined both by in situ and by in vitro formats. Messenger RNAisolated from a test sample can be used in hybridization oramplification assays that include, Southern or Northern analyses, PCRanalyses, and probe arrays. One preferred diagnostic method for thedetection of mRNA levels involves contacting the isolated mRNA with anucleic acid probe that can hybridize to the mRNA encoded by the Homgene. The probe can be a full-length Hom nucleic acid, such as thenucleic acid of SEQ ID NO: 2 or 3 or a portion thereof, such as anoligonucleotide of at least 10 nucleotides in length and sufficient tospecifically hybridize under stringent conditions to Hom mRNA or genomicDNA.

In one format, mRNA (or cDNA prepared from it) is immobilized on asurface and contacted with the probes, for example, by running theisolated mRNA on an agarose gel and transferring the mRNA from the gelto a membrane, such as nitrocellulose. In another format, the probes areimmobilized on a surface and the mRNA (or cDNA) is contacted with theprobes, for example, in a gene chip array. A skilled artisan can adaptknown mRNA detection methods for detecting the level of Hom mRNA.

The level of mRNA (or cDNA prepared from it) in a sample encoded by Homgene can be evaluated with nucleic acid amplification, e.g., by standardPCR (U.S. Pat. No. 4,683,202), RT-PCR (Bustin S. J Mol. Endocrinol.25:169-93, 2000), quantitative PCR (Ong Y. et al., Hematology. 7:59-67,2002), real time PCR (Ginzinger D. Exp Hematol. 30:503-12, 2002), and insitu PCR (Thaker V. Methods Mol. Biol. 115:379-402, 1999), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques known in the art. As used herein,amplification primers are defined as being a pair of nucleic acidmolecules that can anneal to 5′ or 3′ regions of a gene (plus and minusstrands, respectively, or vice-versa) and contain a short region inbetween. Under appropriate conditions and with appropriate reagents,such primers permit the amplification of a nucleic acid molecule havingthe nucleotide sequence flanked by the primers.

For in situ methods, a cell or tissue sample can be prepared andimmobilized on a support, such as a glass slide, and then contacted witha probe that can hybridize to genomic DNA on chromosomes or mRNA thatencodes the Hom polypeptide.

In another embodiment, the methods of the invention further includecontacting a control sample with a compound or agent capable ofdetecting Hom mRNA, or genomic DNA, and comparing the presence of HommRNA or genomic DNA in the control sample with the presence of Hom mRNAor genomic DNA in the test sample.

The above-described nucleic acid-based diagnostic methods can providequalitative and quantitative information to determine whether a subjecthas or is predisposed to a disease associated with aberrant Hom geneexpression, e.g., cancers.

A variety of methods can be used to determine the level of Hompolypeptide. In general, these methods include contacting an agent thatselectively binds to the polypeptide, such as an antibody, to evaluatethe level of polypeptide in a sample. Antibodies can be polyclonal, ormore preferably, monoclonal. An intact antibody, or a fragment thereof(e.g., Fab or F(ab′)₂) can also be used. In a preferred embodiment, theantibody bears a detectable label. The term “labeled”, with regard tothe probe or antibody, is intended to encompass direct labeling of theprobe or antibody by physically linking a detectable substance to theprobe or antibody, as well as indirect labeling of the probe or antibodyby reactivity with a detectable substance. For example, an antibody witha rabbit Fc region can be indirectly labeled using a second antibodydirected against the rabbit Fc region, wherein the second antibody iscoupled to a detectable substance. Examples of detectable substances areprovided herein. Appropriate detectable substance or labels includeradio isotopes (e.g., ¹²⁵I, ¹³¹I, ³⁵S, ³H, or ³²P), enzymes (e.g.,alkaline phosphatase, horseradish peroxidase, luciferase, orβ-glactosidase), fluorescent moieties or proteins (e.g., fluorescein,rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g.,Qdot™ nanoparticles by the Quantum Dot Corporation, Palo Alto, Calif.).

The detection methods can be used to detect the Hom polypeptide in abiological sample in vitro as well as in vivo. In vitro techniques fordetection of the Hom polypeptide include ELISAs, immunoprecipitations,immunofluorescence, EIA, RIA, and Western blotting analysis. In vivotechniques for detection of the Hom polypeptide include introducing intoa subject a labeled anti-Hom antibody. For example, the antibody can belabeled with a detectable substance as described above. The presence andlocation of the detectable substance in a subject can be detected bystandard imaging techniques.

The diagnostic methods described herein can identify subjects having, orat risk of developing, a disease or disorder associated with aberrantHom expression or activity. As described herein, examples of such adisease or disorder include ALL, CLL, AML, and MDS

The prognostic assays described herein can be used to determine whethera subject is suitable to be administered with an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disorder, such ascancer (e.g., ALL, CLL, and AML). For example, such assays can be usedto determine whether a subject can be administered with a cytotoxic drugto treat a cell proliferation disorder or immune-suppressants to treatan immune disorder, including those involved in organ/tissuetransplantation.

Thus, also featured in this invention is a method of monitoring atreatment for a cancer or an immune disorder in a subject. For thispurpose, gene expression levels of Hom can be determined for testsamples from a subject before, during, or after undergoing a treatment.An increase of the expression level of Hom after the treatment indicatesthat the subject can be further treated by the same treatment. Forexample, a patient who has received organ or tissue transplantationoften faces the problems of organ or tissue rejection. That is, the bodyhas an immune response to an organ or tissue which causes failure of thetransplant. To address this problem, organ or tissue transplantation isoften accompanied by nonspecific immune suppression therapy to prevent Tcell-mediated rejection. However, these immunosuppressants can causeinfection, hypertension, cancer, and other undesirable side effects.Therefore, there is a need for monitoring the suppression. To that end,Hom-1 expression level can serve as a marker for a proper level ordegree of immune suppression. A skilled in the art can adjust the amountof immunosuppressants and length of treatment based on the level ofHom-1 expression during the course of the treatment.

Information obtained from practice of the above assays is useful inprognostication, identifying progression of, and clinical management ofdiseases and other deleterious conditions affecting an individual'shealth status. In preferred embodiments, the foregoing diagnostic assaysprovide information useful in progno-stication, identifying progressionof and management of malignancies (cancers) that are characterized bylack or abnormal low level Hom expression. The information morespecifically assists the clinician in designing chemotherapeutic orother treatment regimes to eradicate such malignancies from the body ofan afflicted mammal, e.g., a human.

The specific examples below are to be construed as merely illustrative,and not limitative of the remainder of the disclosure in any waywhatsoever. Without further elaboration, it is believed that one skilledin the art can, based on the description herein, utilize the presentinvention to its fullest extent. All publications cited herein arehereby incorporated by reference in their entirety. Further, anymechanism proposed below does not in any way restrict the scope of theclaimed invention.

EXAMPLE 1 Hom-1, a LEF/TCF-associated Transcription Repressor, is aTumor Suppressor

LEF/TCFs transcriptional factors are the nuclear transcriptionalmediators of canonical Wnt/beta-catenin signaling, which is critical tocell fate determination in early embryogenesis and is implicated as amajor oncogenic pathway in a variety of cancers. LEF/TCFs possess littleintrinsic transcriptional activity, but are tightly controlled byassociated factors. In the presence of Wnt signaling, LEF/TCFs form acomplex with beta-catenin, which drives the expression of LEF/TCFdownstream genes, including well-established oncogenes such as cyclin D1and C-myc. Xom (also known as Vent-2, Xbr-1, and Vox) is a vertebratehomologue of the Drosophila ananassae homeobox gene Om1D. Xom wasidentified as the major downstream mediator of BMP4 and is an essentialconstituent of the ventral signaling center. Similar to beta-catenin,Xom also is an unstable protein. We examined the effect of Xom onLEF/TCF-mediated transcription and identified Xom as a novelLEF/TCF-associated transcription factor.

In this example, through sequence homology searching, distributionprolifing, and functional analysis, we identified a new transcriptcalled Hom-1 as a candidate human Xom homolog. It was found that Hom-1shares the same ORF with the previously known VentX2 but contains a711-bp 5′UTR beyond the known sequence of VentX2. Further assays wereconducted to analyze Hom-1 as described below.

Materials and Methods:

Promoter-Luciferase Assay

For a typical promoter-luciferase assay experiment, 2×10⁵ 293T cellswere seeded onto a 12-well culture plate 24 hours prior to transfection.One microgram of a plasmid encoding gene of interest and 0.3 μgluciferase-reporter construct were mixed with 3 μl of liposometransfection reagent (TRANSIT, Mirus) and transfected into culturedcells following the manufacture's instructions. Forty-eight hours posttransfection, cells were washed with PBS and lysed with a 1× cell lysisbuffer (PROMEGA), scraped, and collected on ice. After a brief spin in atable-top centrifuge, 20 μl of supernatants were mixed with 100 μl ofluciferase assay reagent (PROMEGA), and luciferase activity was measuredusing a TR717 Microplate luminometer (APPLIED BIOSYSTEM).

RNA Isolation, RT-PCR and Real-time PCR

Total RNA was extracted by the TRIzol method. Cells (1×10⁶) werehomo-genized in 1 ml Trisol, and 200 ml chloroform was then added. Aftervortex mixing, the samples were centrifuged at 12,000 rpm, and thesupernatants were collected in new tubes. Isoproponol (500 ml) was addedto each sample, mixed, and kept at room temperature for 30 minutes.Samples were then centrifuged at 12,000 rpm at 4° C. for 30 minutes. Thepellets were collected, washed with 70% ETOH, air-dried, andre-suspended in 20 μl of DEPC-treated H2O. The final RNA concentrationwas determined by OD260 measurement. First-strand cDNA was synthesizedwith the SuperSript First-Strand Synthesis System (INVITROGEN) accordingto the manufacturer's protocol. Briefly, 3 μg of total RNA from eachsample was used for RT-reaction, and 1 μl of the RT product was used forPCR reaction. GAPDH was used as an internal control. The identities ofPCR products were verified by sequencing. Real-time PCR was performedusing the LightCycler System (ROCHE) and the LightCycler FastStart DNAMaster SYBR Green I, according to manufacturer's instructions. Therelative levels of gene expression were calculated by the formula:relative gene expression=2−ΔCd (ΔCd=cycle of the specific gene−cycle ofthe reference GAPDH gene).

Splicing Cloning of Hom-1 and 5′ cDNA Walking

The Hom-1 genomic DNA of 2.4 kb was amplified from a BAC RP13 clonecontaining the Hom-1 genomic sequence with PCR reaction using primers

(SEQ ID NO: 17) F1: 5′ AATTGAATTCAATGCGCCTCTCCTCCTCC 3′, (SEQ ID NO: 18)R1: 5′ TTAATCTAGATCATCAAAATGCATCCCCCGTCTG 3′.

The PCR products were digested with EcoRI and XbaI and cloned into theCS2 vector. The plasmid was transfected into 293T cells. Total RNA fromthe transfected cells was extracted by TriZol methods and subjected toRT reaction using the SuperScript First-Strand Synthesis System(INVITROGEN). The RT products were amplified with primer F1 and R1,digested with EcoRI and XbaI, and then cloned into the CS2 vector.

For 5′ cDNA walking, first-strand cDNA was amplified using total RNAfrom peripheral leucocytes from healthy volunteers. The 5′ un-translatedregion was amplified with forward primers starting at position −100,−161, −291, −357, −471, −711, −843 (the position of the nucleotide “A”of the start codon ATG was designated as position 0).

Immunofluorescence and Co-immunoprecipitation

For immunofluorescence experiments, GFP-Hom-1 and Myc-TCF4 wereco-transfected into HCT116 cells with TransIT (MIRUS). TCF4 was stainedwith anti-myc antibody, followed by secondary Alexa 568 labeled goatanti-mouse antibody (INVITROGEN). Nuclei were visualized with DAPI andimages were taken with co-focal microscopy at the core facility ofChildren's Hospital Boston. For co-immuno-precipitation, affinityprotein A/G beads were prepared by mixing 20 μl of protein A/G agarosebeads (SANTA CRUZ BIOTECHNOLOGY) with 1 μg of the antibodies ofinterest. To prepare the cell lysates, 2×10⁶ of cells were lysed with a1× cell lysis buffer (PROMEGA) containing 1× protease inhibitor reagent(ROCHE). The cell lysates were incubated on ice for 30 minutes, brieflysonicated, and centrifuged at 12,000×g for 5 minutes at 4° C. Thesupernatants were further cleaned by adding 20 μl of protein A/G agarosebeads and 1 μg of pre-immune serum at 4° C. for two hours. After a briefspin, the supernatants were then mixed with 20 μl ofantibody-labeled-protein A/G beads at 4° C. overnight. The beads werewashed four times with PBS 0.2% NP40. Bound proteins were released by 2×sample buffer, boiled at 95° C. for 5 minutes, centrifuged briefly, andsubjected to western blot analysis using specific antibodies asindicated. Mouse anti-myc antibody and goat anti-TCF4 were purchasedfrom Santa Cruz.

Cell Culture, Cell Isolation, and cDNA Array

Cells used in this example include Nalm6 cells, Nalm16 cells, Reh cells,RS11 cells, H Sultan cells, ALL sample cells, CLL sample cells, 293Tcells, Jurkat cells, Tall cells, EBV transformed B cells and matched Bcells from healthy individuals, PC3 cells, LnCap cells, MCF7 cells, MDAcells, SK-N-AS cells, H1299 cells, H460 cells and 116 cells. All thecell lines were maintained in RPMI 1640 or DMEM supplemented with 10%FBS and 1% penicillin and streptomycin. Leucocytes from healthyindividuals were isolated from discarded leukopaks from healthyanonymous blood donors at Children's Hospital Boston. Experiments withhuman materials were performed in accordance with guidelines approved bythe institutional review committee of Brigham and Women's Hospital.

T cells, B cells, granulocytes, and monocytes were isolated with MCASmicrobeads specifically labeled with CD3, CD19, CD15, and CD14antibodies (MILTENYI BIOTEC), following the manufacturer's instructions.PrimeExpress II Human Normal Tissue cDNA Panel (#10020) was purchasedfrom PRIMGEN.

Transient Transfection and Establishment of Hom-1 shRNA Stable CellLines

For HCT116 and 293T cells, all transient transfections were performedwith liposomal TransIT reagent (Mirus) following manufacturer'sinstructions. For Reh and Nalm16 cells, transient transfection wasperformed with electroporation using the Cell Line Nucleofector Kit V(cells) following manufacturer's instructions. Hom-1 shRNA plasmids wereobtained from Origene Technologies; Construct p1: 5′ CAAATCTGCCTGCGCCGGAGAGGACCATG 3′(SEQ ID NO: 10); construct 3: 5′TTCAGAATCGCCGCATGAAACACAAACGG 3′ (SEQ ID NO: 4). To establish Hom-1knock-down cell lines, cells were transfected with Hom-1 shRNA.Forty-eight hours post-transfection, the Hom-1 knock-down cells wereselected with 500 ng/ml puromycin treatment for four weeks.

Cell Viability Assays, MTS Proliferation Assays, and ³H ThymidineIncorporation Assays

For Hom-1 over-expression experiments, cells were transfected withplasmids encoding GFP-Homl or GFP. Twenty-four hours post-transfection,GFP-positive cells were sorted out by FAC G4 Sort Flow Cytometer (BDBIOSCIENCE) and seeded into culture plates.

For cell viability assays, 5×10⁵ cells were seeded into 12-well platesin triplicate. Viable cells were counted four times by Trypan Bluestaining at each indicated time point. Cell viability is presented as apercentage. For MTS proliferation assays, 1×10⁵ cells were seeded into96-well plates in triplicate. Forty-eight hours post-seeding, cellproliferation rate was measured with the Cell Titer 96 AqueousNo-radioactive cell proliferation assay kit (PROMEGA) according tomanufacture's instructions. For ³H thymidine incorporation assays, 1×10⁵sorted cells were seeded into 96-well culture plates in triplicate.Forty-eight hours post-seeding, ³H thymidine (1.0 U^(ci)/well) was addedinto each well for an additional 18 hours. The cells were lysed byfreezing at −70° C. overnight, transferred to membrane, washed with PBSthree times, and subjected to scintillation counting using the TopCountNXT (PACKARD BIOSCIENCE).

CHIP Assay

Hela cells were cultured in 6 cm dishes for 24 hours and thentransfected with constructs encoding myc-Hom-1 or myc-tag. Twenty-fourhours post-transfection, the chromatin immunoprecipitation (ChIP) assaywas performed using an assay kit (Upstate Cell Signaling) followingmanufacture's instructions. Cyclin D1 promoter sequence was amplifiedwith specific primers: F5′-CGGACTACAGGGGAGTTTTGTTG-3′ (SEQ ID NO: 11)and R5′-TCCAGCATCCAGGTGGCGACGAT -3′ (SEQ ID NO: 12), and immunoglobulinheavy chain promoter was amplified with specific primers:F5′-AACCCTTTTCCCCCTCGTCT-3′ (SEQ ID NO: 13), R5′-AGCACCTGTGAGGTGGCTGC-3′(SEQ ID NO: 14). PCR products were analyzed by electrophoresis on 1%agarose gels.

Statistical Analysis

Data were analyzed using the t test. The differences with p value <0.05were considered statistically significant

Results:

Hom-1 is a Xom Homologue

To identify the human homologue of Xom, we used the amino acid sequenceof the Xom homeodomain (HD) as the template to search against the NCBIprotein database. The candidate gene revealed by the search wasdesignated Hom-1 (previously known as Ventx2). The open reading frame ofHom-1 encodes 258 amino acids, in comparison with the 327 amino acids ofXom. As shown in FIG. 1A, the homeodomain of Hom-1 shares 68% identicaland 85% positive (similar) amino acid sequence with Xom. Hom-1 containsan N-terminal serine/threonine-rich domain (aa 4-89), a homeodomain (aa.91-151), and a C-terminal proline-rich domain (aa. 151-258). This issimilar to Xom, which also contains an N-terminal serine/threonine-richdomain (aa. 31-154), a homeodomain (aa. 172-233), and a C-terminalproline-rich domain (aa. 233-326).

Using the Vector NTI protein alignment program, we found that, besidesthe homeodomain, Hom-1 and Xom share strong similarities at the carboxylterminal region and the beginning portion of the amino terminal regionfor about ten amino acids. There is an unaligned portion of theN-terminal region between these two molecules that is also functionallyrelevant. Comparative genomic analysis shows that Hom-1 is preserved inprimates (FIG. 1B), but sequence homology is lost in other species.Besides structural similarities, EST data from CGAP (The Cancer GenomeAnatomy Project) show that the expression pattern of Hom-1 is similar tothe distribution of Xom, both demonstrating very limited expression inadult tissues but expression in embryonic tissues.

To further evaluate the function of Hom-1, its cDNA was obtained throughsplicing cloning, using the BAC RP13 clone as the template. In additionto the known sequence, a novel 5′ untranslated region of 711 bp on thefirst exon of Hom-1 was identified by 5′-cDNA walking.

Xom is a transcriptional repressor of the dorsal-specific geneGoosecoid. To identify any potential functional similarity between Hom-1and Xom, mRNA encoding Hom-1 or Xom was injected into one of the twoblastomeres of Xenopus embryos at the two-cell stage, together with mRNAencoding Activin and the Gsc-luciferase reporter construct. Five embryoswere collected at stage 10, and luciferase activity was measured usingthe PROMEGA luciferase assay system.

It was found that expression of Hom-1 inhibited activin-inducedexpression of the Gsc-promoter, similar to the inhibition ofGsc-promoter by Xom. These results are consistent with the prior findingthat expression of Hom-1/Ventx2 inhibits dorsalization in Zebrafish.Nevertheless, as shown below, Xom transactivates LEF/TCF-mediatedtranscription through its N-terminal domain, which distinguishes Xomfrom Hom-1. Thus, it is concluded that Hom-1 is a Xom homologue ratherthan a Xom ortholog.

Hom-1 Forms a Complex with Lef/Tcf Transcriptional Factors

It was examined whether Hom-1 interacts with LEF1/TCFs. Myc-tagged Hom-1was transiently expressed in HCT116 cells, and potential interactionbetween Hom-1 and TCF4 was characterized by co-immuno-precipitation.When anti-TCF4-coated beads were applied to the HCT116 cell extracts,myc-Hom-1 readily co-immuno-precipitated with TCF4. It was also foundthat Hom-1 co-localized with TCF-4 in a punctate manner in the nuclei oftransfected cells (FIG. 2B). To determine the critical domains of Hom-1involved in the interaction with Lef/Tcf factors, a serial deletionmutants of Hom-1 was made and tested their affinity with TCF4 (FIG. 2A).It was found that if the homeodomain and its surrounding region of 50amino acids were deleted from Hom-1, the resultant mutants did notinteract with anti-TCF4-coated beads by co-immunoprecipitation,suggesting that the Hom-1 homeodomain and its surrounding region playcritical roles in the interaction between Hom-1 and LEF1/TCF factors.

Given that the homeobox domain is a DNA binding domain of Hom-1, 20μg/ml ethidium bromide (EtB) was included to rule out a possibleinterference of nuclear acids in the interaction between Hom-1 and TCF4.It was found that EtB did not interrupt the interaction between TCF4 andHom-1, indicating that the interaction between Hom-1 and TCF4 is notdependent on their association with DNA.

Hom-1 Inhibits Beta-catenin Transactivation of Lef/Tcf-mediatedTranscription

Assays were conducted to examine the effects of Hom-1 on beta-catenintransactivation of LEF/TCFs, using the LEF/TCF reporter TOPflash assay.It was found that Hom-1 alone did not activate LEF1/TCF-mediatedtranscription in 293T cells. Instead, Hom-1 blocked β-catenintransactivation of LEF/TCF-mediated transcription in aconcentration-dependent manner.

To further confirm the inhibitory effects of Hom-1 on beta-cateninsignaling, assays were conducted to examine the effects of Hom-1 onLEF/TCF-mediated transcription in HCT116 cells, which constitutivelyexpress elevated endogenous β-catenin activity. The results show thatHom-1 inhibited LEF1/TCF-mediated transcription in aconcentration-dependent manner in HCT116 cells, but exerted nosignificant effects on the expression of beta-catenin mRNA or protein.

To explore the alternative mechanisms underlying Hom-1 action, it wasexamined whether Hom-1 disrupts the formation of a beta-catenin and TCF4complex, which is required for beta-catenin transactivation of LEF/TCFtarget genes. HCT116 cells were transiently transfected with Myc-Hom-1in increasing concentrations. Forty-eight hours post-transfection,HCT116 cell lysates were collected, and TCF4 was immunoprecipated withspecific antibody. Specific antibodies were used to detect any Hom-1 andbeta-catenin in the immunocomplex. It was found that, with increasinglevels of Myc-Hom-1 in the immunocomplex, the levels of beta-catenin inthe immunocomplex exhibited a corresponding decrease, suggesting thatHom-1 expression disrupts formation of the beta-catenin/TCF4 complex.

Thus, similar to the actions of beta-catenin—associated inhibitors ofbeta-catenin/LEF/TCF signaling such as the Chibby, blocking formation ofa complex between beta-catenin and LEF/TCF factors could explain Hom-1inhibition of LEF/TCF transactivation by beta-catenin.

Hom-1 Modulates the Expression of Beta-catenin/LEF1/TCF Downstream Genes

In this part, assays were conducted to examine the effects of Hom-1 onthe expression of endogenous LEF/TCF target genes, such as cyclin D1.

First, assays were conducted to examine the interaction between Hom-1and the cyclin D1 promoter, using a CHIP assay. A plasmid encodingmyc-Hom-1 and the control myc-tag were transiently transfected into Helacells, which were later subjected to immunoprecipitation with antibodiesagainst myc-tag. The cyclin D1 promoter or IgG heavy chain promoter Cmμ,which was used as a negative control, were amplified with specificprimers. It was found that Hom-1 bound specifically to the Cyclin D1promoter, but not the control Cmμ promoter.

Further, the effects of Hom-1 on the expression of Cyclin D1 weretested, using the cyclin D1 promoter-luciferase assay. Consistent withHom-1 inhibition of LEF/TCF-mediated transcription, it was found thatHom-1 blocked transactivation of the Cyclin D1 promoter-luciferaseconstruct in HCT116 cells, similar to its effect on the TOPflashreporter construct. Consistent with an inhibitory effect of Hom-1 on theexpression of cyclin D1, western blot analysis showed that Hom-1 causeda concentration-dependent decrease in levels of the intra-cellularcyclin D1.

Hom-1-1 is a Negative Regulator of Cell Proliferation

The canonical Wnt/beta-catenin/LEF/TCF pathway has been implicated inmalignant transformation and cell proliferation. In this part, assayswere carried out to assess Hom-1's role in cell proliferation.

The expression of Hom-1 was screened in cancer cells derived from bothsolitary and hematopoietic malignancies by RT-PCR. Hom-1 expression wasidentified only in the Nalm16 lymphoblastic leukemia cells but not othercancer cells (see below and FIG. 4D).

To determine the effect of Hom-1 on cell proliferation, GFP-Hom-1 wastransiently transfected into Reh lymphoblastic leukemia cells, whichdoes not express endogenous Hom-1. The positively transfected cells weresorted with FACS, and the effects of Hom-1 on cell proliferation weredetermined by cell count, MTT metabolic assay, and DNA synthesis ³Hthymidine incorporation assay (FIG. 3A). The results indicated thatHom-1 expression strongly inhibited the proliferation of Reh cells.Similar results were also obtained with Hom-1 on the proliferation ofother cancer cells.

To further evaluate the effect of Hom-1 on cell proliferation, theexpression of Hom-1 was down-regulated in Nalm16 cells using a shRNAtechnique. Four constructs of Hom-1 shRNA were transfected into theNalm16 cells. The effectiveness of these constructs in down-regulatingHom-1 expression were determined by RT-PCR and were further verifiedwith western blot analysis, using a specific Hom-1 antibody raised inthe laboratory. It was found that, while construct 3 had high specificactivity against Hom-1 expression, construct 1 exerted little effect(FIG. 3B) Subsequently, the constructs were used to transfect Nalm16cells, and positively transfected cells were selected by puromycinresistance. The effect of these Hom-1 shRNA on the expression ofendogenous Hom-1 was determined by RT-PCR (FIG. 3B upper panel).Expression of Hom-1 in control and Hom-1 shRNA transfected cells wasfurther determined by immunoblot using Hom-1-specific antibody (FIG. 3Bmiddle panel) and quantified by densitometry (FIG. 3B lower panel). Theresults indicated that construct-3 but not construct-1 effectivelyknocks down the expression of Hom-1 in Nalm16 cells.

The effects of Hom-1 on cell proliferation were further determined bycell proliferation assay and MTT assay. As shown in FIG. 3C, whiledownregulation of Hom-1 with construct 3 was associated withhyper-proliferation of Nalm16, neither the control construct norconstruct 1 had any effect on the proliferation of Nalm16 cells. Theabove results suggested that Hom-1 is a negative regulator of cellproliferation.

To determine whether the effects of Hom-1 on cell proliferation relateto the expression of LEF/TCF target genes, the effects ofdown-regulating Hom-1 on the expression of cyclin D1 was examined. Incontrast to the effect of overexpression, down-regulation of Hom-1 wasassociated with increased cellular levels of cyclin D1 (FIG. 3D). Theabove results suggested that Hom-1 down-regulated cell proliferation atleast partly through inhibiting beta-catenin/LEF/TCF signaling and theexpression of its downstream cell cycle regulators such as cyclin D1.

Hom-1 Expression is Highly Regulated in Hematopoietic Cells andImplicated in Oncogenesis of Lymphocytic Leukemia

To explore the physiological role of Hom-1, its expression wascharacterized in adult tissues, using tissue cDNA array and RT-PCR. Itwas found that the expression of full length Hom-1 was highly restrictedto peripheral blood leukocytes (FIG. 4A). Lineage analysis of peripheralblood leukocytes showed that Hom-1 was expressed in both myeloid andlymphoid lineages, including mononuclear cells, B cells, T cells, andneutrophils (FIG. 4B). Its expression increased as B cells matured (FIG.4C). In contrast to the physiological expression of Hom-1 in peripheralB lymphocytes, Hom-1 was essentially not expressed in cancer cell linesderived from B cell malignancies, except for Nalm16 cells (FIG. 4D).Moreover a screen of cancer cell lines derived from solitary cancersrevealed that Hom-1 was not expressed in cancer cell lines, includingHCT116, SW480, HT29, HepG2, PC3, LnCap, Hela, H460, H1299, MCF7, MDA andSK-N-AS. LEF/TCF factors have been shown to play an essential role inB-lymphocyte development and leukemogenesis. Consistent with Hom-1'srole in oncogenesis of B cell malignancies, the level of full lengthHom-1 expression was significantly reduced during the immortalization ofperipheral B cells by EBV infection.

To further explore the role of Hom-1 in the oncogenesis of B cellmalignancies, the expression of full length Hom-1 was examined inperipheral blood samples from patients with newly diagnosed anduntreated acute lymphoblastic leukemia (ALL) and chronic lymphocyticleukemia (CLL). All CLL samples contained a minimum of 95% CD19+leukemia cells. RT-PCR of the total RNA level from these blood samplesshowed a significant reduction of Hom-1 expression in all seven ALL andten CLL samples (FIG. 5A). It was also found that down-regulation ofHom-1 expression in the peripheral blood of acute and chronic leukemiapatients was associated with an elevation in the expression of cyclin D1(FIG. 5B).

Several mechanisms could explain the highly regulated expression ofHom-1. Besides transcriptional regulation, the Hom-1 transcript containsa long GC-rich 5′ un-translated region (5′UTR) of 711 base pairs. A longatypical 5′UTR is seen in about 10% of mRNAs and often precedes genesthat regulate cell growth and proliferation, such as C-myc, HIF, andLEF/TCF. The long 5′UTR could form a secondary structure to regulatetranslation initiation of Hom-1. Thus, it is possible that Hom-1 5′UTRplays a critical role in regulating the expression of Hom-1 at thetranslational level.

The above results suggested that Hom-1 is a tumor suppressorfunctioning, as least partly, as an inhibitor of the expression ofLEF/TCF-downstream oncogenes such as cyclin D1. The results alsosuggested that Hom-1 can be used for predicting clinical behavior oflymphocytic leukemia and as a target for identifying drugs for treatingvarious disorders, e.g., cancer.

EXAMPLE 2 Hom-1 Induced Apoptosis in p53-Sufficient and -DeficientCancer Cells

Materials and Methods

Cell Culture, Transfection, Chemotherapy

The human colorectal cancer lines HCT116, SW480, and the human lungcancer cell line H460 and H1299 were obtained from America Type CultureCollection (Manassas, Va.). The HCT116 (p53^(−/−)) cell line was a gift.All cell lines were maintained at 37° C. and 5% CO₂. Cell culture mediaincluded McCoy's 5A (INVITROGEN, Carlsbad, Calif.) for HCT116, HCT116(p53^(−/−)) and SW480, RPMI-1640 (INVITROGEN) for H460 and H1299. Thecell culture media were supplemented with 10% fetal bovine serum(HYCLONE, Logan, Utah), 100 units/ml penicillin, 100 μg/ml streptomycin,and 250 ng/ml amphotericin B (MEDIATECH, Hemdon, Va.). Transfection wasdone with Lipofectamine 2000 (INVITROGEN) following the instructions ofthe manufacturer. The anticancer drugs used in the study, including5-fluorouracil (5-FU, 50 ug/ml) and doxorubicin hydrochloride (DOX, 0.4ug/ml) were purchased from SIGMA (ST. Louis, Mo.). All drugs weredissolved in DMSO and diluted to appropriate concentrations with cellculture media.

Plasmids and Construction

A nucleic acid encoding Hom was subcloned by PCR-based technique intothe pCS2+ vector, GFP-tag. The constructs were verified by in vitrotranslation and sequencing.

Confocal Microscopy

HCT116 cells were seeded on glass chamber slides and transfected withHom-GFP expression constructs. Twenty-four hours later, the cells werefixed with paraformaldehyde in PBS, and counterstained by propidiumiodide (PI, SIGMA). After four washes in PBS for 5 min each, the slideswere mounted and analyzed by confocal microscopy.

Apoptosis and Growth Assay

Cells including attached plus floating cells in the medium wereharvested and fixed in a solution containing a final concentration of3.7% formaldehyde, 0.5% Nonidet P-40, and 10 μg/ml4′,6-diamidino-2-phenylindole in PBS. Apoptosis was assessed throughmicroscopic visualization of condensed chromatin and micronucleation. Atleast three independent experiments were carried out for each condition,and a minimum of 400 cells were counted in each measurement.

Cell growth was measured by3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTT) assay in 96-well plates (8,000 cells per well) using the CellTiter96 AQueous One Solution (PROMEGA, Madison, Wis.) following theinstructions of the manufacturer. A_(490 nm) was measured using aVERSAmax Tunable Microplate Reader (Sunnyvale, Calif.). Vehicle-treatedcells were as 1 (100%). Each experiment was done in triplicate andrepeated at least twice.

Colony Formation Assay

Cells were transfected with Lipofectamine 2000 (INVITROGEN) for 24 h in100 mm cell culture dishes according to the instructions of themanufacturer. Then, the cells were collected, and sorted GFP-Vector orGFP-Hom1 positive cells using a FAC G4 sort flow cytometer (BDBiosciences). These GFP-positive cells were plated in six-well plates atdilutions of 1×10⁵ cells each well. Cells were allowed to grow for 5-10days before staining with Crystal Violet (SIGMA). All experiments wererepeated at least twice, and similar results were obtained in eachtrial.

Xenograft Tumors and Tissue Staining

All animal experiments were approved by the Institutional Animal Careand Use Committee at the Harvard Medical School. Cells were transfectedwith Lipofectamine 2000 (INVITROGEN) for 24 h in 100 mm cell culturedishes. Then, the cells were collected, and sorted vector-GFP or Hom-GFPpositive cells using a FAC G4 sort flow cytometer (BD BIOSCIENCES).Xenograft tumors were established by s.c. injection of 1×10⁵ vector-GFPor Hom-GFP positive HCT116 (p53^(+/+)) or HCT116 (p53^(−/−)) cells intoboth flanks of 5- to 6-week-old female athymic nude mice (SIMONSENLABORATORIES, Gilroy, Calif.). At the same time, unsort cell 1×10⁶including GFP-positive and -negative cells also was s.c. injected intoother nude mice for established a unsort xenograft tumors mode. Tumorgrowth was monitored thrice a week by calipers to calculate tumorvolumes according to the formula (length×width²)/2. The Xenograft tumorstissue was immediately fixed in 10% neutral buffered formalin. Thetissues were then embedded in paraffin and sectioned. The sections werestained with hematoxylin and eosin (H&E), and then subjected tohistological analysis. Terminal deoxyribo-nucleotidyltransferase—mediated dUTP nick end labeling (TUNEL) staining was doneusing recombinant terminal transferase (ROCHE, Indianapolis, Ind.) anddUTP-Alexa 594 (MOLECULAR PROBES) according to the instructions of themanufacturers and counterstained by 4′,6-diamidino-2-phenylindole. Allimages were acquired with a Nikon TS800 fluorescence microscope usingSPOT camera imaging software.

Cellular Fractionation

Floating and attached cells were harvested from two 60-cm² dish bycentrifugation, resuspended in homogenization buffer (0.25 mol/Lsucrose, 10 mmol/L HEPES (pH 7.4), and 1 mmol/L EGTA), and subjected to40 strokes of homogenization on ice in a 2-mL Dounce homogenizer. Thehomogenates were centrifuged at 1,000×g at 4° C. for 10 minutes topellet nuclei. The supernatant was subsequently centrifuged at 14,000×gat 4° C. for 30 minutes to obtain cytosolic (supernatant) fractions.

Western Blotting

Total cell lysates, mitochondrial and cytosolic fractions were purifiedand separated by 4-20% Tris-Glycine Gel (INVITROGEN, Carlsbad, Calif.)electrophoresis. For active caspase-3, PARP and Hom analysis, totalcells were extracted and separated by 4-20% Tris-Glycine Gel(INVITROGEN) electrophoresis. Antibodies used include GFP (Santa CruzBiotech, Santa Cruz, Calif.), Histone-1 (Ab-1, NEOMARKERS, Fremont,Calif.), and β-actin (SIGMA), active caspase-3 (BD BIOSCIENCES), andPARP (CELL SIGNALING TECHNOLOGY, Boston, Mass.). Appropriate Horseradishperoxidase-conjugated secondary antibodies were used to detect the boundprimary antibodies antigen complex and developed with Western Lightning®Western Blot Chemiluminescence Reagent Plus (PERKINELMER, Boston,Mass.).

Reverse Transcription-PCR

Total RNA was isolated using the TRIzol® Reagent (INVITROGEN) accordingto the instructions of the manufacturer. First-strand cDNA wassynthesized using Superscript II reverse transcriptase (INVITROGEN).Subsequently, each RT reaction mixture was subjected to PCRamplification with the number of cycles varying from 20 (GAPDH) to 35(Hom). Each cycle consisted of a heat denaturation step (94° C. for 60seconds), an annealing step (57° C. for 30 seconds), and an extensionstep (72° C. for 30 seconds). The PCR products were size fractionated ona 2% agarose gel, and visualized under ultraviolet light. The primersused to amplify Hom included:

(SEQ ID NO: 15) 5′-AAGGCAATTAGGCGCTGCTT-3′ and (SEQ ID NO: 16)5′-ACAGAACAACTGAGTCCTCCA-3′.Statistical Analysis

Results are expressed as mean±SD. Statistical analysis was evaluated byANOVA analysis in which multiple comparisons were performed by using themethod of least significant difference. Differences were consideredsignificant if the probability of the difference occurring by chance was<5 in 100 (P<0.05).

Results

Hom-1 Encoded a Nuclear Protein

Hom-1 is a human homologue of Xenopus Xom and shares similar structurehomology. To identify the intracellular localization of Hom-1, anexpression vector encoding the full-length Hom-1 protein with anamino-terminal GFP tag was constructed. The constructs were transfectedinto HCT116 colon cancer cells, and the intracellular distribution ofthe chimera protein was visualized with confocal microscopy. Nuclei ofthe HCT116 cells were labeled with PI staining. It was found that Hom-1was targeted to the nuclei of transfected cells, where it co-localizeswith PI. To further verify the nuclear localization of Hom-1, sucrosegradient was used to fraction the sub-subcellular compartments beforedetermining the distribution of Hom-1 in each compartment with Westernblot analysis. It was found that Hom-1 was enriched in the nuclearfraction of the transfected cells, while GFP is enriched in thecytoplasmic compartment of the transfected cells.

Hom Suppressed Growth of Human Cancer Cells Through Induction ofApoptosis

As discussed above, Hom-1 is a antagonist of the oncogenic Wntsignaling. To determine the effects of Hom-1 on the growth of solidtumors, assays were carried out to examine the effects of Hom-1 on thegrowth of human cancer cells.

It was found that HCT116 colon cancer cells, H460 lung cancer cells anda human embryonic kidney cell 293T (transformed with adenovirus E1a andcarrying a temperature sensitive T antigen co-selected with neomycin)were transfected with expression vector encoding GFP-Hom-1 or GFP. Forall three cell lines, about 35-50% of the cells were transfected asindicated by the GFP signal. Forty-eight hours after transfection, cellsgrowth was analyzed by MTS assay, following manufacture's instruction.

It was found that GFP did not exert any effects on the growth of testedcells. However, GFP-Hom-1 exerted strong inhibition on the growth oftested cancer cells. Interestingly, the growth inhibitory effects ofGFP-Hom-1 was minimal non-cancer 293T cell line. These findings isconsistent with previous observation named tumor addiction (to oncogenicpathways).

Furthermore, a colony formation assay was conducted to investigate theeffect of Hom-1 on long-term cell survival. HCT116 cells weretransiently transfected with plasmids encoding GFP-Hom-1 or GFP.Forty-eight hours post-transfection, the cells were collected, andsorted by GFP signal, and then plated in six-well plates at a dilutionof 1×10⁵ cells per well. Twenty-four hours post-plating, the HCT116colon cells (90% attached to plate) were visualized with fluorescentmicroscopy and phase-contrast microscopy. Ninety-six hours post-plating,the transfected cells were examined again with phase-contrast microscopyand fluorescent microscopy.

It was found that few GFP positive cells were identified in welltransfected with GFP-Hom-1 at 96 hours post-plating, suggesting thatGFP-Hom-1 suppress the growth of transfected cells. Similar results werealso observed in other human cancer cell lines including Sw480, H460 andH1299. Consistent with the results of transient transfection studies,the growth of 293T cell was only slightly inhibited by GFP-Hom-1. Longterm growth of the sorted transfected cells showed that multiplecolonies formed in GFP-transfected cells, whereas, very few coloniesdeveloped in cells transfected with GFP-Hom-1. There was only 20%reduction in the number of colonies in 293T cells transfected withGFP-Hom-1.

To determine whether this growth suppression was as a result ofapoptosis in cells transfected with GFP-Hom-1, apoptosis was assessedthrough microscopic visualization of the transfected cells to determinethe presence of apoptotic features, such as condensed chromatin andmicronucleation by a Hoechst 33258 staining as previously described(Waldman T, et al. Nature 1996, 381:713). It was found that the GFP didnot induce cellular apoptosis, whereas, the GFP-Hom-1 inducedsignificant amount of cellular apoptosis in human cancer cell lines.Moreover, GFP-Hom-1 did not induce apoptosis in 293T cellular, anon-cancer cell line.

These results suggested that Hom-1 is potent inducer of growthsuppression in human cancer cells, and the effect is partly throughinduction of apoptosis. The growth inhibition effects of Hom-1 issignificantly less in non-cancer human 293T cells, which may reflect theprevious noted phenomena of cancer addiction to oncogenic pathway.

Hom-1-1 Induced Apoptotic Cells Death in p53 Deficient Cells

P53 is a critical tumor suppressor gene. More than fifty percent ofcancers harbor inactive p53 as a result of direct mutations in the p53gene To determine whether the tumor suppression effect of Hom-1 relayson a functional p53, assays were conducted to investigate the effect ofHom-1 on three cancer cell lines, the HCT116 p53KO, p53 null H1299 andp53 mutated SW480, where the p53 function have been lost either as aresult of mutation or artificial knockdown. The methods of analyzing theeffects of Hom-1 on the growth of these cancer cells is essentially thesame as described above.

MTS assays, in vitro cell growth assays, and colony formation assayswere carried out. The results showed that Hom-1 strongly inhibited thegrowth of these tested cancer cells that lack a functional p53 (P<0.01,respectively). In p53 deficient cells, Hom-1 also induced apoptosis asindicated by apoptotic figures.

Hom-1 Induced Caspase-3 Activation

Caspase-3 is a critical executor of apoptosis. It is either partially ortotally responsible for the proteolytic cleavage of many key proteinssuch as the nuclear enzyme poly (ADP-ribose) polymerase (PARP).Caspase-3 has been identified as a key mediator of apoptosis inmammalian cells. Induction of apoptosis leads to cleavage ofprocaspase-3 and the generation of an active 17 kDa caspase-3 and 12 kDacaspase-3 fragments. The activate caspase 3 then targets key modulatorsof the apoptotic pathway including PARP and other caspases.

To investigate whether caspase-3 activation is involved in Hom-mediatedapoptosis, total proteins were extracted, and the caspase-3 activationwas analyzed by western blotting assay using an active caspase-3antibody. It was found that expression of GFP-Hom-1 but not GFPactivated caspase-3 in both p53 sufficient and deficient cancer cells.These results suggested that Hom-1 induced activation of caspase-3 in ap53 independent manner.

Hom-1 Repressed Tumors Growth by Inducing Apoptotic Cell Death In Vivo

To identify whether Hom-1 confers antitumor activity in vivo, bothHCT116 p53 wild-type (WT) cell line and HCT116 p53 knockout (KO) cellline were transfected with expression vectors encoding GFP or GFP-Hom-1.The transfection rate was about 35-40% (FIG. 6A). The growth rates ofthese transfected cells were determined by injecting the cellssubcutaneously in the back of nude mice. The cells expressing GFP wereinjected into the left side of the back, whereas the cell expressingGFP-Hom-1 were injected into the right side of the back. The growths ofthe tumors were examined in situ and as excisions (FIG. 6B).

It was found that GFP-Hom-1, but not GFP, repressed the in vivo tumorgrowth of both p53 sufficient and p53 deficient HCT116 cells. H&Estaining and in situ apoptosis analysis by TUNEL staining showed thattumors expressing Hom-1 demonstrated a large number of fragmented DNA,suggesting that Hom-1 repressed tumor growth in vivo by inducingapoptotic cell death.

EXAMPLE 3 Hom-1 Inhibitor Reduced Expression of Cytokines

Assays were conducted to determine if Hom-1 regulates the expression ofproinflammatory cytokines, e.g., interleukin-1 (IL-1), interleukin-6(IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha),and macrophage colony-stimulating factor (M-CSF), in lymphocytes.Specifically, it was determined whether inhibiting Hom-1 from a cell,via an RNAi agent, would reduce the expression of the cytokines.

Undifferentiated monocyte U937 cells were cultured for four days inmedia including M-CSF, which induces monocyte differentiation. The cellswere electroporated with an RNAi agent directed against either Hom-1(construct 3) or GFP and then cultured for another three days. It wasestimated that only half of the cells electroporated successfullyinternalized the RNAi agent. Lipopolysaccharide (LPS, 1 μg/ml) was thenincluded in the culture media to initiate cytokine expression; the cellswere harvested twenty-four hours later. In the manner described above inExample I, the cells' total RNA was extracted, RT-PCR was performed onthe extracted RNA, and real-time PCR quantified the level of IL-1, IL-6,IL-8, or TNF-alpha mRNA in the cells.

It was found that mRNA levels of the cytokines were greatly reduced inthe cells electroporated with the RNAi agent against Hom-1. The mRNAlevel for each of the cytokines was reduced by from 40% to 60% relativeto the control cells (i.e., cells electroporated with an RNAi agentagainst GFP). As about only half of the cells internalized the RNAiagent, the actual magnitudes of the inhibition were greater than 40-60%and should be 100% or close to 100%.

The results suggested that Hom-1 inhibitors, such as RNAi agents, can beused to reduce the expression of proinflammatory cytokines, therebytreating ARDS and other similar respiratory tract/lung disorders thatare mediated by these cytokines.

EXAMPLE 4 Hom-1 Promoted Lymphocyte Development

Hom-1 expression is highly expressed in normal myeloid and lymphoidlineage cells. Its expression is up-regulated during maturation ofhematopoietic cells.

Undifferentiated monocyte U937 cells containing either GFP^(Tet) orGFPHom-1^(Tet) were used in these experiments. GFP^(Tet) andGFPHom-1^(Tet) are transgenes that are activated when exposed totetracycline (Tet) and thereby overexpress GFP and a fusion of GFP andHom-1, respectively. The cells were cultured in media including Tet for48 hours. LPS was then added as described-above and the cells wereharvested twenty-four hours later. The mRNA levels of IL-1, IL-6, IL-8,TNF-alpha, or M-CSF were determined in the same manner described above.

It was found that the mRNA levels of the cytokines were dramaticallyincreased in cells overexpressing Hom-1. They were about ten to overthree-hundred fold greater than the levels in the control cells, whichoverexpressed only GFP. The results demonstrate that Hom-1 promoteslymphocyte maturation and differentiation.

Further assays were carried out to examine roles of Hom-1 in monocytesand macrophages, which are key players of both innate and adaptiveimmunity. It was found that Hom-1 plays a pivotal role in monocyte tomacrophage differentiation.

More specifically, repressing Hom-1 expression in primary monocytesprofoundly impaired terminal macrophage differentiation. Knockdown ofHom-1 expression in monocytes using the above-described siRNA abrogatedthe morphogenesis of the fibroblast-like shape and substantiallydiminished the expression of cell surface CD71 marker, FcγRI CD64, CD40,CD86, integrins CD11b and CD11c, TLR4 (Toll-like receptor 4), MR(mannose receptor), and CD14. Interestingly, the knockdown did notdecrease the viability of primary monocytes as determined by trypan blueexclusion. The expression of other cell surface molecules, such asHLA-DR, was not affected by the knockdown of Hom-1, ruling out thepossibility that diminished monocyte to macrophage differentiation mayresult from cytotoxicity of Hom-1 suppression. In addition, monocytestransfected with siRNA exhibited reduced phagocytotic activity comparedwith the control cells, suggesting that Hom-1 is required for functionaldevelopment during monocyte to macrophage differentiation

Conversely, over-expression of Hom-1 in monocytes accelerated monocytedifferentiation into macrophages, as indicated by the elevatedexpression of CD71 on the surface of transfected cells. Similarly,ectopic expression of Hom-1 in myeloid U937 cells triggered theirdifferentiation with prominent macrophage features, including expressionof the above-mentioned markers, pronounced morphology changes (cellsbecame adherent and flattened with extensive pseudopodia), enhancedphagocytotic activity, and increased secretion of pro-inflammatorycytokines. The results further showed that M-CSF receptor is a directtranscriptional target of Hom-1.

The fact that Hom-1 expression correlated positively with the expressionof several pro-inflammatory cytokines in clinical patients suggestedthat Hom-1 plays significant role in pathogenesis and treatment ofinflammatory diseases.

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A method for decreasing the level of apro-inflammatory cytokine or for decreasing the level or activity ofinflammatory cells in a subject, comprising administering to a subjectin need thereof an effective amount of an inhibitor of a polypeptidecontaining the sequence of SEQ ID NO: 1, wherein the inhibitor is anRNAi agent containing the sequence of SEQ ID NO:
 6. 2. The method ofclaim 1, wherein the cytokine is TNF-α, IL-1β, or IL6.
 3. A method fortreating a human subject having, or at risk of having, an immunedisorder, comprising administering to a subject in need thereof aneffective amount of an inhibitor of a polypeptide containing thesequence of SEQ ID NO: 1, wherein the inhibitor is an RNAi agentcontaining the sequence of SEQ ID NO:
 6. 4. The method of claim 3,wherein the immune disorder is an inflammatory disorder.
 5. The methodof claim 3, wherein the immune disorder is acute respiratory distresssyndrome (ARDS).
 6. The method of claim 1, wherein the administeringstep is carried out by administering an siRNA including the sequence ofSEQ ID NO:6.
 7. The method of claim 1, wherein the administering step iscarried out by administering an expression construct that expresses ashort hairpin RNA (shRNA) including the sequence of SEQ ID NO:6.
 8. Themethod of claim 3, wherein the administering step is carried out byadministering an siRNA including the sequence of SEQ ID NO:6.
 9. Themethod of claim 3, wherein the administering step is carried out byadministering an expression construct that expresses a short hairpin RNA(shRNA) including the sequence of SEQ ID NO:6.